Abstract

Polymorphisms in the B lymphoid tyrosine kinase (BLK) gene have been associated with autoimmune diseases, including systemic lupus erythematosus, with risk correlating with reduced expression of BLK. How reduced expression of BLK causes autoimmunity is unknown. Using Blk+/+, Blk+/−, and Blk−/− mice, we show that aged female Blk+/− and Blk−/− mice produced higher anti-dsDNA IgG Abs and developed immune complex–mediated glomerulonephritis, compared with Blk+/+ mice. Starting at young age, Blk+/− and Blk−/− mice accumulated increased numbers of splenic B1a cells, which differentiated into class-switched CD138+ IgG-secreting B1a cells. Increased infiltration of B1a-like cells into the kidneys was also observed in aged Blk+/− and Blk−/− mice. In humans, we found that healthy individuals had BLK genotype-dependent levels of anti-dsDNA IgG Abs as well as increased numbers of a B1-like cell population, CD19+CD3−CD20+CD43+CD27+, in peripheral blood. Furthermore, we describe the presence of B1-like cells in the tubulointerstitial space of human lupus kidney biopsies. Taken together, our study reveals a previously unappreciated role of reduced BLK expression on extraperitoneal accumulation of B1a cells in mice, as well as the presence of IgG autoantibodies and B1-like cells in humans.

Introduction

Systemic lupus erythematosus (SLE) is a chronic inflammatory condition with an autoimmune etiology caused by the interplay of several genes and environmental factors. In recent years, many susceptibility genes for lupus have been identified (1, 2). A genome-wide association study found a single nucleotide polymorphism (SNP) in the 5′ upstream region of the B lymphoid tyrosine kinase (BLK) gene associated with SLE (3). Multiple studies have confirmed the association of SNPs in the promoter of BLK with SLE in several populations (4, 5). BLK is also associated with other autoimmune disorders, such as rheumatoid arthritis (6), systemic sclerosis (7), Sjögren’s syndrome (8, 9), primary anti-phospholipid syndrome (10), dermatomyositis (11), and Kawasaki's disease (12). The SNP risk alleles found in regulatory regions have been shown to associate with reduced mRNA levels of BLK and reduced protein expression (3, 13–15).

BLK encodes a nonreceptor member of the Src family of tyrosine kinases. BLK is mainly expressed by B lymphocytes but also, to a lesser extent, by non–B cell lineages, such as plasmacytoid dendritic cells, pancreatic β cells, and γδT cells (13, 16–19). Although Blk phosphorylation is detectable upon anti-IgM stimulation (20–22), BLK expression is downregulated upon BCR stimulation (15), suggesting that BLK may play a dual role downstream of BCR signaling.

Early studies from gene targeted mice showed that the Blk knockout mouse did not have significant phenotypes that would make Blk necessary for B cell activation (23). Revisiting immune phenotypes in the Blk knockout mouse in the C57BL/6 background revealed a role for Blk in the production of higher levels of anti-nuclear Abs, increased B1a cell numbers in the peritoneal cavity, and the presence of hyperresponsive marginal zone B cells (24).

Expansion of B1 cells and their contribution to lupus pathogenesis were reported in several lupus-prone mouse models, and additionally in some mice deficient in genes encoding negative regulators in BCR signaling (25, 26). In mice, B1 cells include B1a (CD5+) and B1b (CD5−) subsets. B1b cells are mainly responsive to T cell–independent Ags, whereas B1a cells can secrete polyreactive IgM natural Abs or even IgG autoantibodies when found extraperitoneally (27–29).

Recently, a population of B1 cells in humans was described in adult peripheral blood and umbilical cord with the CD20+CD27+CD43+CD70− phenotype. These cells have the capacity of stimulating T cells efficiently, producing IgM spontaneously, and show tonic intracellular signaling. They are, in this respect, similar to mouse B1 cells (30). Even though their nature is still a matter of controversy (31–35), this population is expanded in SLE patients (36), whereas it is decreased in human common variable immunodeficiency patients (37).

It is still largely unknown how risk alleles of BLK or its reduced expression promote abnormalities that lead eventually to autoimmunity. We therefore used Blk+/+, Blk+/−, and Blk−/− mice, representing differential expression levels of Blk mRNA and BLK protein (24), and performed a comprehensive analysis of their phenotypes to investigate whether these animals develop any kidney disease. In parallel, we investigated several peripheral blood cell populations of healthy human donors genotyped for the human SNP rs2736340 in the promoter region of the BLK gene (3).

Both in mice and humans, we describe a BLK genotype-dependent increase of B1a and B1-like cells, respectively, and the association with higher levels of IgG anti-dsDNA Abs in serum. We also find immune complex (IC)–mediated glomerulonephritis (GN) in Blk-deficient mice with increased infiltration of B1a-enriched cells into the kidneys. We then make the unprecedented observation of the presence of double-positive CD20+CD43+ B1-like cells in renal biopsies from lupus nephritis patients. Intriguingly, female lupus patients bearing BLK risk alleles had earlier age at onset of lupus nephritis. Our results support a role for BLK in controlling the size of the B1a/B1-like cell pool, the redistribution of B1a/B1-like cells, and the development of lupus nephritis.

Materials and Methods

Mice

Blk knockout mice were a gift from Dr. Sandra Hayes (State University of New York Upstate Medical University, Syracuse, NY). Blk mice were backcrossed 9–10 generations to C57BL/6J mice. All mice were maintained under specific pathogen-free conditions at Oklahoma Medical Research Foundation. All animal procedures were approved by the Institutional Animal Care and Use Committee at Oklahoma Medical Research Foundation.

Human study population and genotyping

Blood samples in EDTA and serum samples were obtained from healthy donors previously genotyped for BLK SNP rs2736340, in the BLK promoter (3), using ImmunoChip (Illumina). For this study 25 CC, 9 CT, and 12 TT allele donors were recruited. Male/female ratios were similar in all the groups (35% for CC, 33% for CT, and 36% for TT). There were no statistical differences in the ages of the different genetic groups (median [interquartile range, IQR] in years, and these were 33 [24–63] for CC, 32 [27–49] for CT, and 34 [28–58] for TT). For some patients only serum samples could be obtained and cytometry data could not be generated. The protocols were approved by the Ethical Committee of the Hospital “Virgen de la Macarena” for the Fundación Pública Andaluza Progreso y Salud in Seville.

Cell preparation, flow cytometry, cell sorting, and Abs

Mice were anesthetized by inhalation with isoflurane. To avoid contamination of cells in kidney blood vessels, perfusion was performed by pumping 10 ml sterile PBS from the left ventricle, whereas the right atrium was opened with a small cut. Perfused kidneys were minced and resuspended in digestion buffer consisting of 2 mg/ml collagenase IV (Sigma-Aldrich) and DNase I (1 μg/ml) in RPMI 1640 complete media and incubated at 37°C for 45 min. Cells were centrifuged, filtered through a 70-μm cell strainer, and mixed 1:1 with 40% Percoll in 1× PBS. This was centrifuged at 3000 rpm for 20 min at room temperature without brake. The loose pellet was washed and cells were counted. Peritoneal lavage was collected after i.p. injection of 8–10 ml sterile 1× PBS.

Single-cell suspensions from mouse spleens, the peritoneal cavity, and kidneys were incubated with fluorescently labeled Abs (detailed in Supplemental Material) after blocking nonspecific binding with anti-mCD16/32 (clone 2.4G2) or 10% rabbit serum (Sigma-Aldrich). Human B1-like cells, B2 cells, and CD19+ B cells were quantified in whole blood by flow cytometry using no wash protocols, immediately after bleeding. Two hundred microliters whole blood in EDTA was treated with the indicated Ab cocktails for 10 min at room temperature in the dark. Afterward, erythrocytes were lysed using Quicklysis solution (Cytognos) following the manufacturer’s recommendations. Data were acquired on an LSR II or FACSVerse flow cytometer (BD Biosciences) and analyzed using FlowJo 9.8 software (Tree Star). Ab-labeled IgMhi and IgMlo/− CD138+ B1a cells from spleens, class-switched B1a cells, and plasma cells from kidneys were sorted by FACSAria (BD Biosciences).

Measurement of Abs by ELISA

For IgG anti-dsDNA ELISAs, 96-well MaxiSorp immunoplates (Nunc) were coated with 500 μg/ml protamine sulfate (Sigma-Aldrich) for 45 min at 4°C and afterward with calf thymus DNA (50 μg/ml) overnight at 4°C. After washing with PBS with 0.05% Tween 20, the plates were blocked for 1 h with 10% calf serum plus 5% goat serum in PBS with 0.05% Tween 20 prior to addition of diluted serum (1:2000 for human samples, 1:25 for Blk mouse samples, and 1:200 for B6.Sle1.yaa mouse samples) for 2 h. Abs were detected using goat anti-mouse IgG-HRP (SouthernBiotech) or goat anti-human IgG-HRP (Life Technologies), and peroxidase reactions were developed using OptEIA tetramethylbenzidine substrate (BD Biosciences). The reaction was stopped using 1 N sulfuric acid and the absorbance at 450 nm was read using a spectrophotometer. In every plate, serum from IgG anti-dsDNA+ B6.Sle1.yaa mice or human lupus patient was included as a reference for normalizing purposes. The arbitrary units were calculated as the ratio OD450 (problem serum)/OD450 (reference serum). The concentrations of total IgG and IgM in sera of genotyped healthy donors were quantified using a human IgG ELISA quantitation kit and a human IgM ELISA quantitation kit, respectively (Bethyl Laboratories).

Mouse histopathology and immunofluorescence staining

Mouse kidneys were fixed in 10% neutral buffered formalin and embedded in paraffin for histological and immunohistochemical analyses. Thereafter, specimens were dehydrated in an ascending ethanol series and embedded in paraffin for subsequent sectioning into 5-7 μm sections using a microtome (Leica). Three or four consecutive renal tissue sections were placed onto a slide for histological analyses. Mouse renal tissue sections were stained with H&E or Jones’ methenamine silver–periodic acid–Schiff according to standard practices.

Two observers blinded to the experimental design evaluated histopathology. The severity of GN was scored as previously reported (40). In brief, renal pathology was evaluated for active proliferative changes in the glomerular mesangium, involvement of the peripheral capillary loops, and extent of inflammatory cell infiltration and was graded on a score of 0 (no pathology) to 4 (maximum pathology). Proliferative/acute GN was calculated as a weighted score as (percentage glomeruli affected × mesangial score) + (percentage glomeruli affected × peripheral score × 2). An average of 149 ± 38 glomeruli was studied for each mouse. Chronic change indicated by glomerular sclerosis, fibrosis, and tubular atrophy was also scored. GN severity results are presented as a sum of proliferative and chronic glomerular pathology.

Mouse kidneys were embedded in 1:1 tissue freezing medium and Tissue-Tek optimum cutting temperature compounds and snap frozen in prechilled 2-methylbutane by dry ice. Then, 10-μm sections were cut on a cyrostat, mounted on Superfrost Plus slides, and fixed at −20°C in acetone for 2 min. After rehydration in staining buffer (1× PBS, 1% goat serum, 2% BSA), slides were stained with the following: IgG (H+L)–Cy3 (Jackson ImmunoResearch Laboratories) and complement 3 (C3)-FITC (Bethyl Laboratories). DAPI counterstain was applied to each slide before coverslip mounting. Images were acquired using a Zeiss Axiovert 200M inverted fluorescent microscope and AxioVision software. Glomerular IgG and C3 depositions were quantified as described (41). In brief, 5 Blk+/+, 5 Blk+/−, and 3 Blk−/− female mice at 1 y of age were used. Five random fields taken under FITC and Cy3 channels of the confocal microscope were subjected to quantification from each mouse. In average, 20 glomeruli from each mouse were graded into three levels, 0–mild, moderate, and severe. After determining the percentage of IgG+ or C3+ glomeruli, individual scores and median percentages of glomeruli were represented in scattered dot and column plots.

Digital RT-PCR and determination of BLK mRNA in CD19+ B cells

Total RNA was isolated from 1 × 106 fresh PBMCs of healthy donors using a High Pure RNA isolation kit (Roche). BLK expression was measured by relative quantification using QuantStudio three-dimensional digital PCR system with probes Hs01017452_m1 for BLK (FAM) and Hs001003268 for HPRT1 (VIC) normalizer using cDNA equivalent to 10 and 20 ng RNA. Fluorescence in QuantStudio three-dimensional digital PCR 20K chips was measured by a QuantStudio three-dimensional digital PCR instrument, and data were analyzed by QuantStudio three-dimensional AnalysisSuite Cloud software (Life Technologies). Because BLK mRNA expression in CD19+ B cells is at least 30-fold higher than in other BLK-expressing leukocytes (13), we have considered that the measured BLK expression comes mainly from B lymphocytes. Thus, the quantity of BLK mRNA measured by digital RT-PCR was estimated using the formula: BLK expression in B lymphocytes = 100 × BLK expression in PBMCs/percentage CD19+ cells in PBMCs. The frequency of CD19+ B cells in the PBMCs was determined by FACS analysis.

Paraffin-embedded specimens from renal biopsies, retrospectively obtained from 13 patients diagnosed with SLE and lupus nephritis at Cliniques Universitaires Saint Luc (Brussels, Belgium) and paraffin tissue sections from a normal adult human kidney (BioChain, catalog no. T2234142) were used for immunofluorescence analyses. Patients were grouped according to the presence of non-risk (CC, n = 6) or risk (TT, n = 7) alleles of SNP rs2736340. Detailed clinical information was recorded, including age, gender, disease manifestations, medical history, and key laboratory parameters. Paraffin tissue sections mounted on slides were deparaffinized and rehydrated sequentially. After Ag retrieval, tissue sections were blocked with 5% normal goat serum (Vector Laboratories) and 5% normal human serum (PAA Laboratories) together in PBS/0.3% Tween 20. Thereafter, CD20 and CD43 Ags were sequentially stained with the specific Abs diluted in blocking buffer. For CD20, the first primary Ab, mouse anti-human CD20 Alexa Fluor 488 (clone L26, eBioscience), was applied on each slide at a 1:50 dilution followed by incubation with the secondary Ab, Alexa Fluor 488 goat anti-mouse IgG (H+L) (Life Technologies), at a 1:400 dilution. This step was performed to enhance the signal of the anti-human CD20 Alexa Fluor 488 primary Ab. After washing slides three times, the second primary Ab, monoclonal mouse IgG1, κ anti-human CD43 (clone DF-T1, DakoCytomation), was applied at a 1:400 dilution followed by the Alexa Fluor 555 goat anti-mouse IgG1 (γ1)–specific secondary Ab (Life Technologies) at 1:200. Nuclei were counterstained using 300 nM DAPI solution (Life Technologies) at room temperature for 5 min. After washing, slides were mounted in Mowiol 4-88 (Sigma-Aldrich) containing 2.5% of the anti-fading reagent p-phenylenediamine (Sigma-Aldrich). Slides were stored in the dark at 4°C. Confocal images were captured using a Zeiss LSM 510 laser scanning microscope equipped with the Zen imaging system. Double immunofluorescence staining was performed at least twice for each patient and the healthy control. Negative control was conducted to ensure that nonspecific staining between the two secondary Abs was absent. For this, immunoreactions were done as described above omitting the mouse IgG1, κ anti-human CD43. For unbiased quantification of CD20+, CD43+, and CD20+CD43+ cells, at least 10 fields were selected in the DAPI channel for each tissue section. The total number of each stained cell type per 0.01 mm2 was calculated (see Table I).

Statistical analysis

Statistical significance between more than two groups was determined by a Kruskal–Wallis nonparametric test with a Dunn multiple comparison test. Statistical significance between two groups was determined by a Mann–Whitney nonparametric test. Graphs and statistical analyses were performed using Prism 6.0 or 5.0 software (GraphPad Software). Values are reported as mean with or without SEM, or median with or without IQR.

Results

A corhort of Blk+/+, Blk+/−, and Blk−/− female mice was monitored for development of anti-dsDNA IgG and lupus nephritis. Sera were collected every 8–12 wk starting at 24 wk through 48 wk of age. Anti-dsDNA IgG levels from Blk+/− and Blk−/− mice started to show a significant increase compared with Blk+/+ mice by week 48, but not earlier (data not shown) (Fig. 1A). However, the level of anti-dsDNA IgG of Blk−/− mice is still much less than that of lupus-prone B6.Sle1.yaa males (Fig. 1A).

Taken together, Blk+/− and Blk−/− mice are prone to spontaneously develop IgG anti-dsDNA Abs. IC-mediated GN developed in female Blk−/− mice at old age.

Splenic B1a cells are increased in female Blk+/− and Blk−/− mice

Two groups of mice were set up for flow cytometry analysis: young mice (≤18 wk old, males and females) and aged mice (≥52 wk old, females only). Total splenocyte numbers were not affected, nor were follicular B, marginal zone B, or transitional B cells (Supplemental Fig. 2 and data not shown). To compare the phenotypes among different Blk genotypes or between young and aged groups of mice correspondingly, we only present data from females in all of the results below. We confirmed an increase of peritoneal cavity B1a (PerC B1a) cells in young Blk−/− mice (Fig. 2A, 2B) using the same gating strategy as previously (24). Interestingly, aged mice had at least 10-fold more PerC B1a cells than did young mice (Fig. 2B). However, enlarged PerC B1a cell pools in young Blk−/− mice disappeared in aged Blk−/− mice (mean values for aged mice: Blk+/+, 35.7; Blk+/−, 70.1; Blk−/−, 51.2; p > 0.05) (Fig. 2B).

Flow cytometry analysis of peritoneal and splenic B1 cell subsets from young and aged female Blk mice. (A) Representative FACS plots of young female Blk mice from the same litter showed the gating strategies to discriminate debris and doublets in total peritoneal cells and for characterization of peritoneal B1a, B1b, and B2 cells. Number represents the percentage within the gated populations. (B) Statistical plots show the absolute numbers of PerC B1a (upper) and B1b cells (lower) in young (8- to 18-wk-old) and aged (52- to 62-wk-old) Blk mice. Data were pooled from seven independent experimental cohorts of young mice and four of aged mice. (C) Representative FACS plots of aged Blk mice from the same litter showed the gating strategies to discriminate debris and doublets in total splenocytes and to characterize splenic B1a and B1b cells. Number represents the percentage within the gated populations. (D) The statistical plots combine the absolute numbers of splenic (SP) B1a cells from cohorts of young (15- to 18-wk-old) and aged (52 to 61-wk-old) female Blk mice showing an increased accumulation of splenic B1a cells (left), but not of splenic B1b cells (right), in aged Blk+/− and Blk−/− mice. Data were pooled from three independent experimental cohorts of young mice and four of aged mice. Both statistical plots in (B and D) are shown as mean with Kruskal–Wallis test (multiple comparison among Blk genotypes) or Mann–Whitney (young versus aged) nonparametric test (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001, ns > 0.05).

B1a cells are only capable of secreting Abs when they migrate to the spleen, bone marrow, or inflamed tissues, but not when residing in the peritoneal cavity (27–29). Because only aged female Blk+/− and Blk−/− mice developed autoimmunity and GN, we first tested whether splenic B1 cells accumulated in those Blk+/− or Blk−/− mice. We adopted FACS analysis strategies for splenic B1 cells from two different laboratories (28, 42, 43) and modified gating strategies to our study accordingly.

Blk+/− and Blk−/− mice have infiltrating B1a cells in the inflamed kidneys

It has been reported that infiltrated B1a cells in inflamed kidneys contribute to nephritis in NZB/W F1 mice (26, 38). We investigated whether the amounts of infiltrated B1a cells in the kidney correlated with the development of nephritis in Blk+/− and Blk−/− mice. Owing to impaired CD23 surface staining from collagenase-treated kidney samples (data not shown), and because we found the highest expression level of β1 integrin/VLA-4 on splenic B1a cells (Supplemental Fig. 3A) among different B cell subsets (Supplemental Fig. 3C), we introduced β1 integrin/VLA-4 as a new marker to characterize B1a cells.

B1a-like cells were characterized as CD19+CD21int/loCD5+CD43+VLA-4+ in splenocytes, regardless of the IgM expression level. Increased splenic B1a-like cells in aged Blk+/− and Blk−/− was confirmed (Fig. 4A). So with confidence, the same parameters were applied to isolated leukocytes from kidneys. The data showed that few B1a-like cells infiltrate the kidneys of young female mice. Instead, an increased percentage of B1a-like cells was recruited into the kidneys of aged mice (Fig. 4B). More importantly, there was significantly increased frequency and absolute numbers of infiltrating B1a-like cells in kidneys from aged Blk+/− and Blk−/− mice, compared with aged Blk+/+ mice (Fig. 4A, 4B). In contrast, infiltration of total CD19+ B cells was not affected by Blk genotypes in aged mice (Fig. 4C). The data demonstrated that B1a cell recruitment into inflamed kidneys is enhanced due to reduced or lost Blk expression. In agreement with this finding, the increase of infiltrating B1a cells into kidneys correlated with lupus nephritis development in aged females.

We next examined whether we would observe a similar phenomenon in healthy carriers with risk alleles of the human BLK gene known to associate with reduced mRNA of BLK. Allele T of SNP rs2736340 in the BLK promoter region is associated with SLE risk and with low expression of BLK mRNA (3). In agreement with previous reports (3, 13), carriers of the CC allele showed relatively higher mRNA expression levels of BLK than did CT- or TT-risk allele carriers (Fig. 5A). According to Griffin et al. (33) and recommended modifications (31, 32), we characterized human peripheral blood B1-like cells as CD19+CD3−CD20+CD43+CD27+ (Fig. 5B). Most B1-like cells are CD70− (94.5%, Fig. 5B) as described in Griffin et al. (30). Consistent with highly expressed VLA-4 on murine B1a cells, most human B1-like cells also expressed higher levels of VLA-4 than did B2 cells (CD19+CD20+CD43−) (Supplemental Fig. 3D). Importantly, TT-risk allele carriers had increased percentages of B1-like cells in PBMCs, compared with donors with CT and CC genotypes (Fig. 5C). There was an inverse correlation between BLK mRNA expression and B1-like cell frequency (Fig. 5D). Although no difference between genotypes was detected in total IgM and IgG Abs in serum (Fig. 5E), we observed a significant increase in the level of IgG anti-dsDNA Abs in CT- and TT-risk allele carriers, compared with CC allele carriers, even when they were still lower than those detected in the sera of SLE patients (Fig. 5E). Taken together, healthy individuals bearing BLK TT-risk allele have more B1-like cells in peripheral blood and higher levels of IgG anti-dsDNA Abs in serum associated with low expression of BLK mRNA as compared with individuals bearing CT and CC alleles.

We analyzed biopsies from 13 SLE patients with lupus nephritis for the presence of B1-like cells in inflamed kidneys (Table I). CD20 and CD43 were used as markers in the immunofluorescence staining of paraffin kidney sections, and nuclei were stained with DAPI for better visualization. CD20−CD43+ (T cells) and CD20+CD43− cells (B cells) were detectable in the human kidney biopsies, suggesting the presence of T and B cells, respectively, in the tubule or interstitium (Fig. 6). CD20+CD43+ double-positive cells (yellow on merged images) were also observed and considered as B1-like cells. CD20+CD43+ B1-like cells accumulated mostly in the interstitium, but less (or fragmented) in the tubule, and they were perfectly distinguishable from CD43 single-positive T cells or CD20 single-positive B cells (Fig. 6). The areas where CD20+CD43+ B1-like cells were observed had clear foci of infiltration. Cells were not observed in the glomeruli. No B1-like cells were detected in kidney biopsies of healthy donors (Fig. 6). Therefore, to our knowledge, this is the first demonstration of the presence of B1-like cells (CD20+CD43+) in human lupus nephritis biopsies. We quantified the numbers of CD20+ B cells, CD43+ T cells, or CD20+CD43+ B1-like cells per 0.01 mm2 in each immunofluorescently stained slide and the values are shown in Table I. We did not observe differences in B1-like cell infiltration between BLK non-risk or risk allele carriers with nephritis (Table I). Nevertheless, we observed a tendency of female SLE patients bearing BLK risk alleles to have an earlier age of nephritis onset (not SLE onset) as compared with those bearing BLK non-risk alleles (median value [IQR]: 24.0 [19.8–33.0] versus 41.5 [33.8–45.3], p = 0.020, Mann–Whitney U test) (Table I).

Presence of B1-like cells in the kidneys of SLE patients with lupus nephritis. Immunofluorescence for CD20 (green) and CD43 (red) was conducted to determine the presence of B1-like cells (CD20+CD43+ double-positive) in SLE patients with lupus nephritis and a healthy control. In all patients examined, B1-like cells were found regardless of their BLK genotype. The staining of four representative patients and one healthy control is shown. B1-like cells (arrows) present in the interstitium show a strong CD20+CD43+ staining in the plasma membrane whereas B1-like cells in the tubule (dashed line) show weaker staining, which is more dotted. In the interstitium, B1-like cells were mostly found in nests of single CD20 and CD43 cells, respectively (patients 4, 12, and 13). We did not observe cells inside glomeruli, but instead in clear foci of infiltration. The signal in the tubule varies from very strong (patients 6, 12, and 13) to almost absent (patient 4). Patients 4 and 6 are non-risk allele carriers, whereas patients 12 and 13 are risk allele carriers. Scale bars, 10 μm.

Discussion

We describe that reduced genotype-dependent expression level of BLK in mice and humans clearly correlated with the increased presence of B1a cells in mice and with a similar increase of B1-like cells in humans. Moreover, we observed a clear increase of IgG anti-dsDNA Abs in healthy individuals with BLK risk genotypes, particularly those homozygous for the risk alleles. In the mouse, we were able to follow the development of kidney disease. We observed that Blk-deficient mice showed a later onset kidney disease, and they had an increased infiltration of B1a cells into kidneys, prior or close to the appearance of histological kidney inflammation. Furthermore, aged Blk+/− and Blk−/− female mice had increased serum IgG anti-dsDNA Abs, IgG IC, and C3 depositions, supporting the development of lupus nephritis. In humans, we observed the presence of B1-like cells in kidney biopsies of lupus nephritis patients. Probably owing to the small numbers of individuals and different stages of the renal disease, we were unable to detect a relationship between the presence of B1-like cells and BLK genotypes. Nevertheless, we did find a potential relationship with earlier onset of lupus nephritis. In such a case, BLK risk variants, added to other susceptibility genes involved, might contribute to earlier development of renal nephritis. The data also support prior studies of genetic association between lupus nephritis and risk alleles of BLK (45, 46) using the same SNP we used in the present study.

The most striking phenotype we observed in aged Blk+/− and Blk−/− mice was the IC-mediated GN. Hybrid strains between 129 and C57BL/6 mice, widely used to generate gene-targeted mice, are spontaneously predisposed to development of humoral autoimmunity with low levels of GN (47). Blk mice that we used were backcrossed to C57BL/6 for nine generations and the Blk gene is located in chromosome 14, but not chromosomes 1, 4, or 7, which contain several loci of lupus susceptibility genes (48, 49). Importantly, we did observe our aged Blk+/− or Blk−/− mice showing typical autoimmune phenotypes, such as IC-mediated GN and anti-dsDNA IgG production with a gender bias, but complete absence of autoimmune disorders in Blk+/+ littermates. This clearly indicated that the contribution to autoimmunity is derived from reduction or loss of Blk.

Samuelson et al. (17, 24) reported that reduced expression of Blk enhanced nephrosis in B6.lpr/lpr mice, but failed to observe any obvious autoimmune symptoms in the B6.Blk+/− mice. The aged group of female mice in our report is 1 yr old, but they used 5- to 6-mo-old mice. Moreover, our data showed that anti-dsDNA IgG Abs were not detectable until 48 wk of age. Therefore, age is one of the critical elements for Blk+/− and Blk−/− mice to get signs of lupus.

B1a cells are polyreactive or even autoreactive with low affinity to Ags (28, 50). We confirmed the presence of increased PerC B1a cells in young female Blk−/− mice (24). The role of PerC B1a cells in lupus pathogenesis is largely unknown, because PerC B1a cells do not produce Igs (28, 29). Previous work has shown that serous cavities are reservoirs of B1 cells and that B1 cells can recirculate to secondary lymphoid organs and tissues (51, 52) to differentiate into natural IgM (44) or even IgG-producing cells, for instance, upon infection (27). Even natural IgM is produced from steady-state B1 cells in spleen and bone marrow (53). Thus, we argue that splenic B1a cells, but not PerC B1a cells, may contribute to lupus pathogenesis. Our data suggest that accumulation of B1a cells in the spleen and their ability to then differentiate into IgG-secreting cells in this proper microenvironment in Blk+/− and Blk−/− mice could be a plausible mechanism to initiate autoantibody production, followed by the initiation of lupus-related symptoms.

Recent reports have shown a high frequency of autoantibody-secreting cells and long-lived CD138+ plasma cells within inflamed kidneys of NZB/W F1 lupus mice (26, 38). Furthermore, class-switched autoreactive B1a cells in the course of murine lupus or type 1 diabetes are subsequently accumulated in the inflamed target organs (29, 54). We reasoned that infiltrated B1a cells produce more autoreactive IgG Abs in the kidneys of Blk+/− and Blk−/− mice, just as B1a cells did in the spleen. Surprisingly, we were unable to detect reasonable production of IgG Abs by class-switched CD138+ B1a cells and CD138+ plasma cells isolated from the kidney with appropriate controls (Supplemental Fig. 4B, 4E). Owing to abundant IgG production from CD138+ splenic B1a cells, this suggests the possibility that IgG autoantibodies that deposited in the kidney may be produced and recirculated by Ab-producing cells from the spleen. Alternatively, the infiltrated B1a-like cells in the inflamed kidneys may have different fates. These will need to be further investigated.

The appearance of autoantibodies secreted by autoreactive B cells is thought to play a role as initiator or executor in autoimmune pathogenesis (54, 55). Our data clearly indicate that reduction of Blk expression increases the accumulation of splenic B1a cells from an early age and CD138+ IgG-producing B1a cells in the spleen at old age in our mice, possibly supporting the reasons why the development of the disease occurred at a late age in these mice. We noticed that B1a and B1-like cells express higher levels of VLA-4 (α4β1 integrins) (Supplemental Fig. 3C, 3D), and this has been reported also for CD43 (Figs. 3, 5) (56) and CD9 (57). Those adhesion molecules might either help B1a or B1-like cells to be retained in the peritoneal cavity or the spleen or promote their migration into, and egress from, lymphoid organs (52). Highly expressed VLA-4 on B1a cells can bind to VCAM1 on follicular dendritic cells (fDCs), supporting a close interaction between B1a cells and fDCs (43, 58). It further suggests that autoantibody production from fDC-associated splenic B1a cells, a T-independent type of B cell activation (58), would act as initiator to form autoantigen-containing ICs. Then those autoantigen-containing ICs displayed on fDCs (59, 60) may activate emerging autoreactive B2 cells and trigger T-dependent germinal center responses.

To our knowledge, ours is the first observation that CD20+CD43+ B1-like cells are found in the tubulointerstitial space of human lupus nephritis kidneys. The same distribution has been observed for infiltrating CXCR3+CD4+ T cells (61) or plasma cells (62) known to induce GN. In fact, we also observed CD43+ T cells and CD20+ B cells in the interstitium. How the infiltrated B1-like cells collaborate with different recruited immune cells to trigger nephritis is still largely unexplored. Previous data derived from the NZB/W F1 lupus-prone mice (26, 38) and our current observations from aged Blk+/− and Blk−/− mice suggest that B1-like cells could have a pathogenic role in the development of lupus kidney disease in humans.

Our research has identified the cellular alterations by which low BLK-expressing polymorphic variants contribute to lupus, with particular emphasis on the potentially pathogenic role of B1 cells. The new understanding of B1a/B1-like cells and their contribution to autoimmunity could have broad implications in the regulation of immune responses in different diseases, from autoimmune diseases to infections.

Disclosures

Funding from the Innovative Medicines Initiative to members of the PRECISESADS project (M.E.A.-R., B.L., N.V., I.G., A.D.-B. and C.M.) includes in-kind or cash contributions from pharmaceutical companies to the institutions involved (Université Catholique du Louvain and the Fundación Pública Andaluza Progreso y Salud which is part of the Andalusian government and administers GENYO). The other authors have no financial conflicts of interest.

Acknowledgments

We thank the team of the Oklahoma Medical Research Foundation Image Core Facility, Oklahoma Medical Research Foundation Flow Cytometry Core Facility, and Biorepository and Pathology Shared Resource Facility at Icahn School of Medicine at Mount Sinai for technical support. We also thank Dr. Ryuji Iida for discussion of FACS analysis of B1 cells and Dr. Lori Garman for technical guidance for the ELISPOT assay. We thank Ashley Bell, Marina Kravtsova, Huining Da, and Farideh Movafagh for taking care of the mouse colonies and their genotyping, and Maria José Luque for processing human samples.

Footnotes

This work was supported by funding primarily obtained from Centers of Biomedical Research Excellence/National Institutes of Health Project GM103456-10, the Alliance for Lupus Research, Instituto de Salud Carlos III Grants PI12/02558 and PI10/0552 (partly supported by the European Fund for Economic and Regional Development), and by the Fundación Ramón Areces. This work has also received support from the European Union/European Federation of Pharmaceutical Industries and Associations Innovative Medicines Initiative Joint Undertaking (“Re-classification of Systemic Autoimmune Diseases” Project) under PRECISESADS Grant 115565.